This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Direct cell-cell communication as mediated by gap junctions has been shown repeatedly to be a necessary component of homeostasis and is highly regulated during the cell cycle, developmental processes and cell proliferation. Connexin diseases result when gap junction proteins mis-traffic or mis-function and loss of gap junction intercellular communication is concomitant with carcinogenesis. Mitosis, an integral part of the cell cycle, causes significant morphological and biochemical changes throughout the entire cell. Certainly, some of the biggest changes in GJ structure morphology and distribution occur as tissue culture cells internalize their GJs as they go into mitosis and undergo cell rounding and cytokinesis. In unstimulated (normally trafficking cells) NRK cells, Cx43 isolated from immunoprecipitated cell lysates show three bands on Western blots. These include a nonphosphorylated form (NP) and two phosphorylated forms (P1 and P2) that are predominately phosphorylated on multiple, unidentified serine sites. GJ plaques contain predominantly the P1 and P2 forms while the NP form is localized intracellularly. Using immunofluorescence, nocodazole synchronized mitotic cells display an entirely intracellular localization of Cx43. The Cx43 found in these mitotic cells is uniquely phosphorylated and migrates as a distinct P3 species in a p34cdc2/cyclin B kinase-dependent manner. Most connexins contain sites for multiple sites for phosphorylation. These phosphorylation sites are important for proper protein trafficking, assembly and degradation and likely play a role in signal transduction as these connexins contain several protein kinase consensus phosphorylation sequences. This project is focused on determining whether recycling occurs during mitosis, correlation with serine phosphorylation events and where old versus new protein is found in the cell during mitosis, using the NCMIR tetracysteine technology to do so.